Biochemische und molekularbiologische Charakterisierung des Riboflavintransports in Ashbya gossypii

Förster, C.

Dissertation / PhD Thesis/Book

Biochemische und molekularbiologische Charakterisierung des Riboflavintransports in Ashbya gossypii

Förster, C. (Corresponding author)FZJ*

1999
Forschungszentrum, Zentralbibliothek Jülich

Jülich : Forschungszentrum, Zentralbibliothek, Berichte des Forschungszentrums Jülich 3626, (1999) = Hannover, Univ., Diss., 1999

Please use a persistent id in citations: http://hdl.handle.net/2128/4425

Report No.: Juel-3626

Abstract: The filamentous fungus Ashbya gossypii is used for industrial riboflavin (vitamin B2) production. Biosynthesis, regulation and production parameters have been studied thoroughly in the past, resulting in weil established fermentation processes with a reported maximum yield of 15 g/l. The present work adresses now the still outstanding analysis of riboflavin transport processes in A. gossypii. On the basis of this characterization, producing strains with an improved riboflavin excretion were developed. Ouring fed-batch fermentation processes as weil as in its natural habitat, A. gossypii frequently encounters hyperosmotic stress which leads to reduced riboflavin oversynthesis. The osmoadaptation mechanisms of A. gossypii were investigated, hereby distinguishing between halo- and osmotolerance by exposure to NaCI and mannitol stress. Growth and ultrastructure of differently treated cells were compared and the intracellular contents of compatible solutes and inorganic ions were quantified. Glycerol was found to be the predominant compatible solute in Ashbya gossypii, accumulation of inorganic ions could not be detected. Measurement of glycerol uptake under isosmotic conditions as weil as upon hyperosmotic stress revealed the existence of a highly active glycerol uptake system, which however was down-regulated under hyperosmotic stress. Investigation of glycerol biosynthesis by measuring glycerol-3-phosphate-dehydrogenase activity under hyperosmotic conditions indicated that accumulation of glycerol in A. gossypii is solely due to biosynthesis. Riboflavin uptake and excretion at the plasma membrane could be characterized to be mediated by two separate carrier systems. Hereby, highly active export opposes uptake of very low activity, rendering re-uptake of excreted riboflavin negligible for netto-excretion. Riboflavin production by A. gossypii starts in the late growth phase when septa are formed in the hyphae and vacuoles become visible. Vacuoles begin to pool big amounts of riboflavin. As the vacuolar compartmentation of metabolites in general plays a major role in the regulation of metabolism in fungi, determination of riboflavin fluxes across the tonoplast was crucial for understanding riboflavin transport in A. gossypii. For this purpose, two different approaches to prepare and characterise vacuoles from the filamentous fungus were developed, Le. the isolation of vacuoles from hyphal cells and the controlled permeabilisation of the plasma membrane. Furthermore, a purification procedure for the vacuolar H+-ATPase of A. gossypii was established to characterise its subunit composition, hydrolytic activity and sensitivity against specific inhibitors. Using these cell biological tools, dependence of vacuolar riboflavin accumulation on the activity of the V-ATPase could be proven. By transport assays, dynamic compartmentation of riboflavin between cytosol and vacuole was shown to be based on active, ATPase-dependent accumulation and an energy-independent efflux mechanisrTJ. By inhibition of the V-ATPase with the specific inhibitor concanamycin A, effective redirection of vacuolar riboflavin contents into the growth medium was achieved. In order to construct new strains with impaired vacuolar riboflavin accumulation, the vacuolar ATPase subunit A structural gene VMA 1 of A. gossypii was cloned and disrupted. Cloning was achieved by PCR using degenerated oligonucleotide primers derived form conserved sequences of the Vma1 proteins from yeast and filamentous fungL Oisruption of the Ag VMA 1 gene led to complete excretion of riboflavin into the medium instead of retention in the vacuolar compartment, as observed under concanamycin A inhibition. Interestingly, in contrast to S. cerevisiae where disruption of the VMA 1 gene is conditionally lethai and to N. crassa, where viable disruptants could not been isolated, disruption of the VMA 1 gene in A. gossypii did not cause alethal phenotype. The new disruptant strains only show significant riboflavin transmembrane fluxes via the plasma membrane export carrier system. They could therefore be apt for screening on the plasma membrane export carrier to achieve further improvement of riboflavin excretion in the future. As an alternative strategy to impair vacuolar riboflavin accumulation, the 2- dimensional separation of vacuolar membrane proteins via 2-D-gelelektrophoresis was established as a screening method on the vacuolar riboflavin uptake protein via spot matching analysis between strains which differ in their vacuolar riboflavin accumulation.

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